PROJECT 2 PROJECT SUMMARY: Fanconi Anemia (FA) is an inherited DNA repair disorder characterized by congenital abnormalities, cancer predisposition, and progressive bone marrow failure. FA is caused by biallelic mutations in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway. Although the precise biochemical functions of the FA/BRCA pathway remain unclear, the pathway promotes homologous recombination (HR) repair. Due to the underlying DNA repair defect, FA cells are hypersensitive to genotoxic DNA crosslinking agents. The mechanism of the bone marrow failure (BMF) in FA remains elusive. Our recent studies suggest that BMF results, at least in part, from increased p53 expression in hematopoietic stem and progenitor cells (HSCPs), leading to progressive cell cycle delay and apoptosis. BMF may also result from the accumulation of DNA damage from the endogenous crosslinking agent, acetaldehyde, and the selective toxicity of this agent to hematopoietic stem cells. Recently, we identified hyperactive TGF? signaling as a mechanism of bone marrow suppression in FA. Disruption of TGF? signaling, through the use of shRNAs, sgRNAs, and small molecule inhibitors confirmed the suppressive role of the pathway on FA cell growth. We hypothesize that an upstream inhibitor of the TGF? pathway (i.e., a monoclonal antibody to TGF? itself) will inhibit this pathway and rescue the function of the HSPCs, resulting in an increased probability of rescuing bone marrow function in FA patients. It is possible that TGF? inhibitors may also promote the clonal evolution of premalignant or malignant hematopoietic stem cell. The specific aims of Project 2 are: 1) determine the mechanism by which TGF-? inhibitors promote FA cellular growth and regulate DNA repair, 2) to determine whether inhibition of TGF-? pathway rescues hematopoietic defects in FA mouse models, and 3) to determine whether inhibition of TGF-? pathway rescues hematopoietic defects in primary bone marrow cells from FA patients. Project 2 will interact extensively with Project 1 (Grompe), which will analyze other small molecules capable of improving FA cell growth and Project 3 (Shimamura), which will analyze the effect of these small molecules on primary human FA cells and will provide additional preclinical data for an FA clinical trial. The projects will utilize four Core programs, as described in the accompanying Core components.